Safety

Experiments were conducted in the Undergraduate lab of the Dept. Of Biotechnology at IIT Madras. The lab cohered to the standards for a bio-safety 1 lab according to the Center for Disease Control and Prevention,Atlanta. Bio-safety Level 1 is suitable for work involving well-characterized agents not known to consistently cause disease in immunocompetent adult humans, and present minimal potential hazard to laboratory personnel and the environment. BSL-1 laboratories are not necessarily separated from the general traffic patterns in the building. Work is typically conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is not required, but may be used as determined by appropriate risk assessment techniques. Laboratory personnel must have specific training in the procedures conducted in the laboratory and must be supervised by a scientist with training in microbiology or a related science.

Contents 1 Standard Microbiological Practices 2 Safety Equipment (Primary Barriers) 3 Laboratory Facilities (Secondary Barriers) 4 Simple Questions

Standard Microbiological Practices

1. Statement of Purpose (SOP) have been developed and are kept for easy access which enlists all the safety measures each experiment should be abided for.
2. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures and specimens are in progress.
3. People wash their hands after handling viable materials, after removing gloves, and before leaving the laboratory.
4. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. People who wear contact lenses in laboratories must wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.
5. Mouth pipetting is prohibited .Instead mechanical pipetting devices are to be used.
6. Policies for the safe handling of sharp instruments are clearly instructed.
7. All procedures are performed carefully to minimize the creation of splashes or aerosols.
8. Work surfaces are decontaminated atleast once a day and after any spill of viable material.
9. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method for example Autoclaving. Materials to be decontaminated outside the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. These materials are packaged in accordance with applicable local, state, and federal regulations before removing from the facility.

Safety Equipment (Primary Barriers)

1. Special containment devices or equipment such as a biological safety cabinet is generally not required for manipulations of agents assigned to Bio-safety Level 1.
2. It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.
3. Gloves are must especially if there is a cut on the hand or if a rash is present. Alternatives to powdered latex gloves should be available.
4. Protective eyewear should be worn while conducting experiments in which splashes of microorganisms or other hazardous materials are anticipated.

Laboratory Facilities (Secondary Barriers)

1. Laboratory has doors for access control.
2. A sink is provided in the Laboratory for washing hands.
3. The Laboratory is designed such that it can be easily cleaned.
4. Bench tops are impervious to water and are resistant to moderate heat, organic solvents, acids, alkalis, and chemicals used to decontaminate the work surface and equipment.
5. Laboratory furniture is capable of supporting anticipated loadings and uses. Spaces between benches, cabinets, and equipments are accessible for cleaning.
6. It is made sure that the Laboratory is cut-off from the external environment.
7. We have an accesible compartment in the lab for MSDS(Materials Safety Data Sheet) where in we have a roster of all the chemicals and what needs to be done in case of accident.

1. Would any of your project ideas raise safety issues in terms of:

• researcher safety,
• public safety, or
• environmental safety?

Researcher Safety

• Chassis: For ensuring researcher safety, we have chosen E.coli as our chassis. They are part of the normal flora of the human gut, and can benefit their hosts by producing Vitamin K2,and by preventing the establishment of other pathogenic bacteria within the intestine.
• EtBr: Ethidium bromide is an intercalating agent and is/was commonly used as a fluorescent tag (nucleic acid stain) for agarose gel electrophoresis in our lab. Ethidium bromide is thought to act as a mutagen because it intercalates double stranded DNA (i.e. inserts itself between the strands), deforming the DNA. This could affect DNA biological processes, like DNA replication and transcription. Hence, we do not wash the gel in EtBr solution. Instead, EtBr is added in minimal concentration to the agar solution before it solidifies. It is strictly observed that gloves are used when handling EtBr or EtBr-containing gel. The gels are discarded considering them to be 'Hazardous Waste'.
Public Safety
• All work with live E.coli cells is carried out in the laminar hood that eliminates the possibility of any GMO(Genetically Modified Organisms) from being released out.
• Laboratory has been provided with doors, as these GMO should not be readily accessible to unintended personnel.
• It is made sure that appropriate measures for waste disposal are taken. Waste is not washed down the sink or thrown into public dust-bins.
• Environment Safety
• All contaminated waste is treated as 'Bio-Hazardous Waste' and is decontaminated before being put into a red trash bag or a sealed medical waste box. The bio-hazard bag is placed in a pre-chosen area for pick up.
• We believe extensively in the three 'R's - Reduce, Reuse and Recyle. By considering these constraints, we have learnt to plan our experiments better.

No. All bio-bricks made are according to the safety guidelines provided by the Center for Disease Control and Prevention.

Yes, our project is supported by the Dept. of Biotechnology, IIT Madras. They have provided us with Biosafety level 1 facilities for all our experimental work.

Antibiotic resistance has been a public health problem of increasing magnitude, and finding effective solutions to address this problem is a critical focus of CDC activities.

1. Decreasing the susceptibility of recombinant cells to antibiotics proves to be a high risk to the environment.
2. Our new idea is to use proteorhodopsin construct as a tool for selecting transformed mutants. We are designing a new plasmid that does not contain antibiotic resistant gene. Instead it will have our synthetic construct to serve the same purpose. We are introducing a positive control as the cell growth is aided rather than hindered as in the case of antibiotics. We envisage that this make antibiotic based screening technique obsolete.

References

• Cupillard, L. et al., 2005. Impact of plasmid supercoiling on the efficacy of a rabies DNA vaccines to protect cats. Vaccine 23: 1910-1916.
• JM Tiedje et al., 1989. The planned introduction of genetically engineered organisms: Ecological considerations and recommendations Ecology, 70(2), 1989, pp. 298-315